| Bovine mastitis occurs frequently in dairy cows and is often caused by various aetiological organisms,especially Escherichia coli and Staphylococcus aureus.Lipopolysaccharide(LPS)and lipoteichoic acid(LTA)are key virulence factors of Escherichia coli and Staphylococcus aureus respectively,and both of them could cause inflammatory reaction in the mammary glands.In healthy mammary glands,alveolar epithelial tight junctions(TJs)constitute the blood-milk barrier,which prevents milk components from leaking from mammary gland alveolars into the blood serum.Bacterial mastitis causes pathogen-dependent damage of the blood-milk barrier and is accompanied by changes in the tight junction permeability in bovine mammary epithelial cells(BMECs).The aim of this study is to investigate the global transcriptional response variations by RNA-seq to analyse BMECs which were stimulated with LPS and LTA to identify specific proinflammatory factors that play important roles in blood-milk barrier damage during E.coli-induced and S.aureus –induced mastitis.In this study,we first used BMECs as pattern recognition receptors and stimulated them with LPS or LTA to investigate the global transcriptional response variations of BMECs through RNA-Seq analysis.Then,we identified specific proinflammatory factors that would play important roles in blood-milk barrier damage during mastitis caused by E.coli and S.aureus by analyzing differentially expressed genes(DEGs).Among the DEGs,interleukin-1β(IL-1β)was selected because its mRNA expression was increased significantly by LPS,its enrichment was involved in multiple inflammatory signal pathways.Furthermore,its roles in blood-milk barrier damage during the process of mastitis were investigated.To validate our RNA-seq results,we selected some differentially expressed genes randomly and IL-1β was selected to analyze their expression by quantitative real-time PCR(qRT-PCR).We analyzed mRNA and protein expression of IL-1β in mastitis tissue and normal tissue from bovine mammary glands by qRT-PCR and ELISA to confirm that IL-1β did participate in the process of inflammation in mammary gland.We further used exogenous IL-1β treating BMECs to simulate the effect of IL-1β on the tight junction permeability of mammary epithelial cells and the mechanism.Besides,IL-1β-induced decline in BMEC proliferation under the treatment condition was also observed in this study.We used Western Blotting(WB)and immunofluorescence to analyze the expression of three tight junction proteins,ZO-1,occludin and claudin-1 in BMECs to further explore the effect of IL-1β on tight junction permeability of BMECs,finding that IL-1β treatment significantly decreased the expression of ZO-1,occludin and claudin-1 in protein level compared with those in the non-treated group.Furthermore,we verified IL-1β-induced increases in the tight junction permeability between BMECs by trans-epithelial electrical resistance(TEER),analyzing the levels of FITC-dextran across the monolayers,and the number of neutrophils that migrated through the IL-1β-treated BMEC monolayers.In tissue level,IL-1β-induced damage of blood-milk barrier in mice mammary tissue was found by hematoxylin-eosin staining,IF,and the ultrastructure of the mammary glands with transmission electron microscopy.To investigate whether the process was regulated by myosin light chain kinase(MLCK),we used the MLCK inhibitors ML-7 pretreating BMECs in these experiments.We found the pretreatment of ML-7 could make BMECs tight junction permeability declined.We further used ERK1/2 inhibitors treating BMECS and detected the expression of protein and mRNA of MLCK by WB and qRT-PCR,illustrating IL-1β-induced increase in tight junction permeability of BMECs was via the IL-1β-ERK1/2-MLCK axis.These results provided a comprehensive analysis of gene expression profiles elicited by LPS and LTA in BMECs,and IL-1β could have effects on BMECs tight junction and blood-milk barrier through IL-1β-ERK1/2-MLCK axis pathway.Our data provide insights into the functions of IL-1β in blood-milk barrier damage caused by mastitis in dairy cows,contributing to the protection and therapy of bovine mastitis. |
